PEG2000-DBCO surface coating increases intracellular uptake of liposomes by breast cancer xenografts

Given our interest in the utility of liposomes for molecular imaging and theranostics, we investigated how coating the outer layer of the liposome affects internalization by breast cancer cell lines in vitro and in breast tumor tissues in vivo. Indeed, we discovered that a remarkably high liposomal uptake can be achieved by DBCO (dibenzocyclooctyne) soft coating. Our data demonstrates that decorating the terminal lipid with a DBCO moiety at a specific density induces increased tumor uptake in vivo (tumor uptake ~ 50%) compared to conventional undecorated liposome (tumor uptake ~ 20%). In this study, we report improved visualization of breast cancer cells in vivo using a 4T1 orthotopic breast cancer model and primary breast tumor xenograft models MDA-MB-231 and MDA-MB-436. L-PEG2000-DBCO coated liposomes demonstrate increased accumulation in breast cancer cells independent of tumor size, type, position, receptor expression, as well as the condition of the host mice. We expect these findings to have a major positive impact on the practical utility of liposomes in image-guided applications and precision medicine theranostics.

Liposomes are lipid vesicles consisting of a lipid bilayer encapsulating an aqueous core and are considered among the most promising and effective drug-delivery vehicles 1,2 . Liposomes have been extensively studied over the past three decades to optimize their clinical potential. Among the most successful applications of liposomes in drug delivery are the two liposomal doxorubicin formulations (Doxil, Myocet) approved for clinical use in ovarian cancer and multiple myeloma, among other diseases [3][4][5] . However, despite these therapeutic successes, the clinical development of liposomes in molecular imaging and in image-guided theranostics is substantially farther behind. So far, the main limitation to overcome is the relatively low liposomal uptake by tumor tissues. There is an unmet need for new liposomal formulations that can facilitate high tumor uptake in order to facilitate clinical translation of these amazing nanocarriers.
Surface modifications enable the custom design of liposomes for diagnostic, therapeutic, and image-guided delivery applications [6][7][8][9][10][11] . The unique advantages of liposomes include minimal immune reactivity, reduced proteolytic degradation, increased circulation times and reproducible assembly in a cost-effective manner. As a result, liposomes are almost magical nanocarrier tools for diagnosis, monitoring and management of human disease 12,13 .
Given our interest in the clinical development of liposomes for molecular imaging theranostics, we investigated how coating the outer layer of the liposome at a specific surface density affects cellular internalization in vitro and in vivo. Indeed, we discovered that remarkably high liposomal uptake by tumor tissues can be achieved in vitro and in vivo by DBCO (dibenzocyclooctyne) soft coating. Our data shows that decorating the terminal lipid with a DBCO moiety at specific density produces profound tumor uptake in vivo (~ 50%) compared to the traditional undecorated liposome (tumor uptake ~ 20%). In an animal model, we were able to visualize increased uptake by 4T1 orthotopic breast cancer cells and xenografts from primary breast cancer cell lines MDA-MB-231 and MDA-MB-436. Our findings are consistent with recent findings that interactions between the liposomal surface and the cell membrane significantly influences cellular uptake. Better understanding of how the liposomal surface regulates cell internalization pathways presents an opportunity for improved intracellular drug delivery 14  www.nature.com/scientificreports/ Our findings will pave the way for the development of the next generation of liposomes with modified surface properties that facilitate efficient tumor uptake and, in turn, demonstrate immense potential for clinical use in precision medicine theranostics. We expect to harness the advantages of these enhanced liposomes for precision image-guided surgery, precision detection of tumor with positron emission tomography (PET) radiotracers and tumor treatment via precise delivery of radiotherapeutic payload to the primary tumor and its distant metastases. These ambitious studies are ongoing in our laboratory and will be published in due course.

Results
Liposome assembly. The liposomes were assembled as illustrated in Fig. 1a following these steps: skeleton lipids (DOPC), functional lipids (DSPE-PEG 2000 -DBCO, or DSPE-PEG 2000 ) and imaging materials (DiI, or DiR) were dissolved into chloroform and formed the thin film followed by rehydration, extrusion and dialysis. The size and zeta potential were ~ 95 nm and − 4.8 mV respectively (Table S1). L-PEG 2000 -DBCO surface morphology was imaged using scanning electron microscopy (SEM) as shown in Fig. 1b. L-PEG 2000 -DBCO (L-DBCO) diameter was approximately 96 nm with 25 Å distance between adjacent DBCO terminals ( Fig. 1b and Table S1). Liposomal uptake in small high-grade neoplasia foci. We utilized optical imaging in vivo to further confirm and build on the in vitro data above. We tested the performance of L-PEG 2000 -DBCO and L-PEG 2000 in a small orthotopic tumor (15 ~ 25 mm 3) to mimic the metastatic foci or the relapse foci after surgical resection 16,17 . The tumor growth and the metastatic spread of 4T1 cells in BALB/c mice was reported to mimic stage IV human breast cancer 18 . Indeed, the data shown in Fig. 3a-c demonstrates superior tumor uptake of L-PEG 2000 -DBCO compared to L-PEG 2000 liposome. The L-PEG 2000 -DBCO exhibits remarkably high uptake in tumor foci, as high as 54%, while liver uptake was limited to 16%. In contrast, the L-PEG 2000 failed to detect this small tumor with 77% the liposome entrapped by liver and the ER system (Fig. 3c,d). The accumulation rate of the two liposomal formulations in tumor tissues is illustrated in Fig. 3c,d. Overall, the L-PEG 2000 -DBCO displayed 700% increased tumor accumulation over the L-PEG 2000 .

Liposomal uptake in normal and breast cancer cells by flow cytometry and confocal micros-
Moreover, a second liposomal administration after 96 h did not cause accelerated blood clearance. It has been reported repeated administration PEG-conjugated substances including PEGylated liposomes can cause immunogenic response resulting in the increased clearance and reduced efficacy of PEG-conjugated substances/ PEGylated nanocarriers [19][20][21] .  Fig. 6a,b. It is important to note, that the small tumor is implanted in the fat pad and therefore it was hard to isolate from the para-cancer tissue. As a result, the tumor size in Fig. 6b appears to be larger than the accurate size of the solid tumor as was measured by the caliper (photo is shown in Figure S4). Also, Fig. 6b indicates that our L-PEG2000-DBCO has a super targeting capacity on both cancer tissue and para-cancer tissue as the signal of para-cancer tissue is shown in Fig. 6b. Furthermore, L-PEG 2000 -DBCO was superior to L-PEG 2000 in tumor xenograft with larger size as shown in Fig. 6b. Ex vivo optical imaging also confirmed the in vivo data as shown Fig. 6c.

Discussion
Despite significant work on liposomal technology over the last several decades, approaches to optimize the surface formulation of liposomes have remained largely unexplored. Our findings demonstrate a key role for surface functional moieties in cellular internalization and tumor uptake. Liposomal surfaces have been extensively utilized to conjugate drugs (the so-called targeted liposome) for various therapeutic applications, some using the DBCO moiety as the site for drug conjugation through copper free "click chemistry" [22][23][24] . However, surface conjugation may reduce the efficiency of liposomal cellular internalization and tumor uptake thus reducing  (Fig. 5d). We expect our findings to find application in the efficient delivery of molecular imaging compounds, imageguided probes, and cancer therapeutics. One advantage is the ability of L-PEG 2000 -DBCO to visualize small tumors such as a small orthotopic 4T1implant (Fig. 2). L-PEG 2000 -DBCO was able to detect a small subcutaneous breast tumor (MDA-MB-231, size 10 ~ 20 mm 3 ) which was invisible to both the naked eye and the bright field as indicated by arrows in Fig. 6. Taken together, the data in Figs. 3 and 6 demonstrate that L-PEG2000-DBCO has shown the capacity to visualize the small tumor in both allogeneic (4T1) and xenogeneic (MDA-MB-231) transplantation models. Furthermore, L-PEG 2000 -DBCO was able to detect, visualize and discriminate between a small tumor (10 ~ 15 mm 3 ) and the adjacent main MDA-MB-231 tumor (196 ~ 255 mm 3 ) and to accumulate in the small tumor with the same signal density of that of the large tumor ( Figure S3). Therefore, we are currently pursuing the utility of L-PEG 2000 -DBCO for molecular imaging and image-guided surgery.
Future perspective. The mechanism that drives the high tumor uptake of L-PEG 2000 -DBCO and how this is facilitated by the DBCO moiety is not fully understood. For example, conjugation of L-PEG 2000 -DBCO with DV1-N3 peptides leads to diminished tumor uptake, similar to L-PEG 2000 , underscoring the key role of the DBCO moiety in driving high tumor uptake 12 . It is likely that the enhanced permeability and retention (EPR) effect contributes to enhanced tumor uptake. Long circulation times allow the liposome to penetrate preferentially into tumor tissue through permeable tumor vasculature and to remain in the tumor bed through impaired www.nature.com/scientificreports/ lymphatic drainage 25 . However, the EPR effect alone was reported to offer less than a twofold increase in nanodrug delivery 25,26 . Further studies of DBCO-labeled liposomes will be needed to help unravel the mechanism of high accumulation of L-PEG 2000 -DBCO in tumor tissues and low accumulation in non-target tissues that ordinarily mediate therapeutic toxicity. Enhanced chemical modifications of DBCO-labelled liposomes may lead to enhanced formulations with improved tumor specificity. These studies are ongoing in our laboratory and will be reported in future publications. In summary, the liposome surface may significantly influence in vitro cellular uptake and in vivo penetration into tumor tissues while minimizing penetration into off-target tissues. We are optimistic that our findings will pave the way for the design of next generation liposomes for efficient delivery of molecular imaging and imageguided probes as well as anti-cancer therapeutics.

Conclusion
We performed a series of in vitro and in vivo experiments to demonstrate that liposomal surface modification may play a key role in inducing high cellular internalization in vitro and high liposomal accumulation in cancer tissues in vivo. Specifically, we discovered that a soft layer of DBCO induces a remarkable increase in tumor uptake of L-PEG 2000 -DBCO compared to L-PEG 2000 despite both liposomal formulations sharing an identical skeleton. We have demonstrated the increased ability of L-PEG 2000 -DBCO to accumulate in breast cancer foci independent of tumor size, type, position, receptor expression, as well as the condition of the host mice. Remarkably, a significant reduction in L-PEG 2000 -DBCO uptake in the liver and off target tissues was also observed compared to L-PEG 2000 . Altogether, our findings will pave the way for the development of new liposomal formulations with enhanced internalization by tumor tissues. We are currently exploring the utility of L-PEG 2000 -DBCO as an effective carrier of molecular imaging and image-guided probes.  www.nature.com/scientificreports/ studies. All cancer cell lines were cultured in DMEM with 10% FBS and 100-unit penicillin-streptomycin. All cells were maintained at 37 °C in a humidified incubator with 5% CO 2 .
Liposomal cellular uptake studies. For liposome binding analyzed by flow cytometry, Vero, MCF-7, MDA-MB-231, and MDA-MB-436 (2 × 10 6 ) were seeded in a 75 cm 2 flask for 2-5 days. After reaching 50% confluence, the cells were detached by 0.25% trypsin/0.1% EDTA followed by washing with PBS twice. After blocking with BSA (1%) for 30 min, samples were stained with liposomes with DiI dye for 2 h at 37 °C in a humidified incubator with 5% CO 2 (0.15 mM of lipids per 10 6 cells). After washing with PBS twice, the samples were resuspended in 500 μL PBS and evaluated by flow cytometry using a BD LSR II Analyzer (B&D Bioscience, CA). Liposomal uptake in cells was analyzed by confocal microscopy. MDA-MB-231 cells (2 × 10 5 ) were seeded in a Lab-Tek II Chamber Slide System separately with 2 mL medium overnight at 37 °C. Samples were stained with liposomes with DiI dye for 2 h at 37 °C in a humidified incubator with 5% CO 2 (0.15 mM of lipids per 10 6 cells). After the medium was removed, cells were rinsed with PBS twice and fixed with 4% formaldehyde in PBS at RT for 10 min. DAPI was used to stain the cell nucleus followed by washing with PBS three times. Cells were examined under a LSM 710 Confocal fluorescent microscope (Zeiss). Digital images were captured and processed with software Image J (NIH). Optical in vivo and ex vivo imaging. In vivo and ex vivo imaging was performed using IVIS Lumina III system (Perkin Elmer). Briefly, to make sure the fluorescence intensity at the same level, 3 × 100 μL of the L-PEG 2000 and L-PEG 2000 -DBCO solutions were placed into 96 well plates separately before administration and scanning under DiR near infrared filed via the following parameters: excitation filter 740 nm, emission filter 790 nm, binning 4 or 8, f/Stop 2. These mice administrated the different liposomes were scanned non-invasively three times under anesthesia (2% isoflurane via the vaporizer of the IVIS instrument). After the in vivo analysis, mice were sacrificed immediately. The organs (tumor, liver, heart, lung, spleen, brain, kidney, small intestine, and colon) were collected, and images were acquired using the same parameters as described above. Collected images were analyzed using Living Image 4.3.1 software (Perkin Elmer): ROIs were designed in order to appropriately select each organ and radiant efficiency calculated.
Cryostat sectioning. The tumor bearing mice were injected the L-PEG 2000 and L-PEG 2000 -DBCO liposomes with 2% DiI components, respectively. After 24 h of liposome administration, the tumors and livers are harvested and frozen into − 80 °C refrigerator. The frozen samples were further cut to Sections (10 μm) with a cryostat (CM3050 S, Leica, Germany). The sections were stained with DAPI, followed by sealing with mounting oil, and then observed using a confocal laser scanning microscope (Zeiss, LSM 710).

Statistical analysis.
All quantitative experiments were run in triplicates and the results were expressed as mean ± standard deviation, unless indicated otherwise. Statistical analysis of the data was performed by two-way analysis of variance (ANOVA) with Tukey's post-test. Differences between groups at a level of p < 0.05 were considered statistically significant (*represented) and those at p < 0.01 as highly significant (**represented).

Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files]. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.